Naturally occurring uranium primarily consists of three isotopes in the following percentages (by weight): .sup.238 U 99.283%; .sup.235 U 0.711%; and .sup.234 U 0.005%. Enriched uranium, produced for nuclear power generation and weapons manufacture, contains greater then 0.711% .sup.235 U. Uranium containing less than 0.711% .sup.235 U is considered "depleted" uranium. Depleted uranium usually contains less than 0.3% .sup.235 U and is therefore less than half as radioactive as natural uranium. Because it is extremely dense, depleted uranium has several applications including the armor plating of military vehicles. It is also used in armor piercing munitions that were first used extensively in the Persian Gulf War. Unfortunately several military personnel were injured with internalized depleted uranium particles in this conflict. This was a medical concern because of the known health risks associated with enriched uranium. Research shows that internalized uranium is eliminated from the body in the urine via the kidneys (H. C. Hodge, Uranium, Plutonium, Transplutonic Elements: Handbook of Experimental Pharmacology, Springer-Verlag, Berlin, Vol 36 (1973), pp. 5-68). However, at the time, there was not a rapid and convenient method to measure depleted (or natural) uranium in biological fluids (urine in particular) that was suitable for a battlefield situation. Rapid assessment of the presence of depleted uranium in injuries is necessary for proper treatment decisions.
Previous art shows a variety of techniques for the measurement of uranium in biological fluids. Chakavarti and colleagues (1980, Int. J. Appl. Radiat. Isotop., 31, pp. 793-795) teach that the amount of .sup.235 U in urine can be determined by the fission track etch technique. This technique was improved on by Ide et al. (1979, Health Phys., 37, pp. 405-408) and called the delayed neutron emission method. Addition advancements in this area led to the technique of neutron activation analysis (1992, H. S. Dang, V. R. Pullat, and K. C. Pillai, Radiat. Protect. Dosim., 40, pp. 195-197). While these techniques are sensitive, they require extensive, labor-intensive sample preparation, as well as a nuclear reactor to provide the thermal neutrons needed for sample activation.
U.S. Pat. No. 4,198,568 to Robbins and Kinrade (1980) discloses that uranium determination in aqueous samples is achieved by ultraviolet light-induced phosphorescence of the uranium. Zook, Collins, and Pietri teach that uranium in biological samples can be determined by the method of pulsed laser-induced fluorescence (1981, Mikrochem. Acta, II, pp. 457-468). After further technical improvements (1992, R. Brina and A. G. Miller, Analyt. Chem., 64, pp. 1413-1418), the method of laser-induced kinetic phosphorimetry was developed (1995, R. Brina, American Laboratory, May, pp. 43-47). However, these methods are hindered by the presence of quenching substances in the sample (1989, E. S. Gladney, W. Moss, M. A. Gautier, and M. G. Bell, Health Phys., 57, pp. 171-175). This results in lower sensitivity or a requirement for extensive sample preparation before analysis. In addition, the instrumentation necessary for these analyses severely restricts their use in a field situation.
The technique of inductively coupled plasma mass spectrometry is also used to determine uranium content in biological samples (1989, E. S. Gladney, et al., Health Phys., 57, pp. 171-175; 1996, Z. Karpas, et al., Health Phys., 71, pp. 879-885). Goldstein and coworkers (1997, Health Phys., 72, pp. 10-18), as well as McKibbin (U.S. Pat. No. 5,190,881), teach that alpha-spectrometry can also be used to measure uranium content in biological fluids. However, inductively coupled plasma mass spectrometry requires instrumentation that would be difficult to maintain in a field situation and the technique of alpha-spectrometry is hindered by the extensive sample preparation time needed.
For use in the field, spectrophotometric or colorimetric detection methods are preferable. Fitoussi, Lours, and Musikas (U.S. Pat. No. 4,349,350) teach that uranium in an organic solvent can be added to a mixture of a neutral organophosphorus compound and a dialkyl dithiophosphoric acid. This results in a complex whose optical density can be determined at 390 nm. While this application can be used to determine uranium levels from organic extracts obtained from the reprocessing of nuclear fuels, there is no indication that it can be used for biological fluids such as urine. Furthermore, considering the extensive sample preparation required, this method could not be conveniently used in a field situation. That is also the case for the dye arsenazo III which has been described for uranium determination in organic-solvent extracts of samples (L. C. Baylor and S. M. Stephens, U.S. Pat. No. 4,424,211) and reversed-phase column chromatographically purified geological samples (1988, X. Wu and W. Qi, Analyt. Chim. Acta 214, 279-288). The stain 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol has been used to detect uranium in organic solvent-extracted geological samples (1971, D. A. Johnson and T. M. Florence, Analyt. Chim. Acta 53, 73-79; 1972, P. Palkalns and B. R. McAllister, Analyt. Chim. Acta 62, 207-209) or kerosene-diluted uranium leach liquors from nuclear fuel reprocessing facilities (1979, S. J. Lyle and M. Tamizi, Analyt. Chim. Acta 108, 267-275; 1985, E. A. Jones, Analyt. Chim. Acta 169, 109-115; 1990, S. D. Hartenstein, Analyt. Chim. Acta 228, 279-285). These methods have never been tested with uranium-containing biological samples, such as urine. They also require extensive sample preparation before the uranium levels can be determined. In addition, extraction of biological samples with organic solvents of the type used for geological samples would result in phase separation. Because any uranium in a biological sample could be bound to lipids (1969, D. O. Shah, J. Colloid Interface Sci. 29, 210-215) and proteins (1995, B. Volesky and Z. R. Holan, Biotechnol. Prog. 11, 235-250), as well as to inorganic components, organic extraction of the biological sample would partition the uranium between the organic and aqueous phases. Organic solvents also cause the precipitation of proteins. Therefore organic extraction of a protein-containing biological sample would result in three components: an organic phase, an aqueous phase, and a protein precipitate. All three of these components are capable of containing significant amounts of uranium. Thus, for an accurate determination of the total amount of uranium present in the sample, all three components (organic phase, aqueous phase and protein phase) would have to be analyzed. Therefore, the application of the current methods of uranium (depleted or natural) determination to a battlefield situation is severely hindered by one or both of the following factors:
(a) Extensive sample preparation is required before analysis. Extraction into organic solvents, column purification, and acid-digestion of samples at high temperatures are examples of some of the preparatory procedures necessary for current uranium detection methods. PA1 (b) Many of the uranium determination procedures need elaborate instrumentation. Nuclear reactors, inductively coupled plasma mass spectrometers, and laser-induced kinetic phosphorimetric analyzers are not commonly found in battlefield medical facilities. PA1 (a) to provide a process for the detection of uranium, both natural or depleted, in biological fluids including, but not limited to, water, urine, blood serum, saliva, amniotic fluid, cerebrospinal fluid, sweat, stool extract, synovial fluid, tears, semen extract, sputum, and peritoneal fluid; PA1 (b) to provide a process for uranium determination that can be conducted rapidly and accurately; PA1 (c) to provide a process for uranium determination that does not require extensive sample preparation before analysis; PA1 (d) to provide a process for uranium determination that does not require the use of complicated instrumentation; PA1 (e) to provide a process for uranium determination that requires only a visible-range spectrophotometer or colorimeter for instrumentation; PA1 (f) to provide a process for uranium determination that can be used in a battlefield situation; and PA1 (g) to provide a process for uranium determination that requires little or no technical training to conduct.